The removal of selected sub-fragments of foreign DNA introduced into plant cells or plants, but which have subsequently become obsolete or even unwanted, for various reasons, after introduction thereof, has been the subject of intensive research. Examples of such sequences are e.g. selectable marker genes which were necessary for the isolation of transgenic plants but which are no longer required in the mature plants. Methods to achieve efficient elimination thereof mostly rely on site-specific recombination or transposition (see e.g Hohn et al. (2001) Curr. Opin. Biotechnol. 12: 139-143).
Siebert and Puchta (Plant Cell (2002) 14: 1121-1131) described that transgenic sequences flanked by sites of a rare cutting restriction enzyme can be excised efficiently from the genome of a higher eukaryote by homologous recombination as well as by non-homologous end-joining.
WO03/004659 relates to recombination systems and to a method for removing nucleic acid sequence from the chromosomal DNA of eukaryotic organisms. The document also relates to transgenic organisms (preferably plants), containing the described systems or produced by the described methods.
WO05/090581 relates to constructs and methods for eliminating maker sequences from the genome of plants, based on dual-function selection marker which—depending on the employed compound—can act as both negative and counter-selection marker.
However the described methods mostly require the use of an in vitro culture method to identify or select those plant cells in which the deletion of the DNA sequences to be removed has effectively taken place and to regenerate a plant from such cells.
US patent application 2005/0060769 proposes a method to prepare a recombined transgenic Zea mays plant or plant cell from a first transgenic Zea mays plant cell, wherein the transgene in the recombinant plant or plant cell has an altered genetic structure relative to the genetic structure of the transgene in the first transgenic plant cell, due to homologous recombination-mediated transgene deletion.
WO97/30166 or U.S. Pat. No. 6,407,314 describe promoter fragments from a microspore-specific gene from tobacco that can be used for expression of genes in microspores.
WO06/105946 has described methods and means for the efficient removal of a selected part of a DNA sequence of interest previously introduced into the plant without resorting to in vitro culture during the removal step. The method described therein relied on the expression of a double stranded DNA break inducing rare cleaving endonuclease under the control of a microspore specific promoter. The microspore specific promoter which was specifically disclosed was the promoter region from the NTM 19 gene in tobacco as described in WO97/30166.
Provided herein are alternative methods and means for the removal of such a selected part of a DNA sequence as described hereinafter in the different detailed embodiment, examples, figures and claims.
Another problem that has been solved by the present invention concerns the targeted and exact exchange through homologous recombination of a target DNA sequence in a cell of a plant for a replacement DNA sequence without leaving footprints of the procedure, and without having to resort to in vitro culture methods after the initial step of homology recombination. To this end, the herein described methods for efficient removal of selected subsequence of a part of a DNA molecule previously inserted in the genome, preferably the nuclear genome of cells of a plant, through intrachromosomal homologous recombination can be conveniently used.
The need to control the site of transgene integration in plants has been recognized early on, and several methods have been developed in an effort to meet this need (for a review see Kumar and Fladung (2001) Trends in Plant Science, 6: 155-159). These methods mostly rely on homologous recombination-based transgene integration, a strategy which has been successfully applied in prokaryotes and lower eukaryotes (see e.g. EP0317509 or the corresponding publication by Paszkowski et al. (1988) EMBO J. 7: 4021-4026). However, for plants, the predominant mechanism for transgene integration is based on illegitimate recombination which involves little homology between the recombining DNA strands. A major challenge in this area is therefore the detection of the rare homologous recombination events, which are masked by the far more efficient integration of the introduced foreign DNA via illegitimate recombination.
One way of solving this problem is by selecting against the integration events that have occurred by illegitimate recombination, such as exemplified in WO94/17176.
Another way of solving the problem is by activation of the target locus through the induction of double stranded DNA breaks via rare-cutting endonucleases, such as I-SceI. This technique has been shown to increase the frequency of homologous recombination by at least two orders of magnitude using Agrobacteria to deliver the repair DNA to the plant cells (Puchta et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93: 5055-5060).
WO96/14408 describes an isolated DNA encoding the enzyme I-SceI. This DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.
WO00/46386 describes methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell through I-SceI double strand break. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.
Chilton and Que (Plant Physiol. (2003) 133: 956-965) and Tzifira et al. (Plant Physiol. (2003) 133: 1011-1023) report that T-DNA preferentially integrates in double stranded DNA breaks, artificially induced by the rare-cleaving enzymes I-SceI or I-CeuI. The reports also included donor T-DNA vectors which comprised a recognition site for the respective rare-cleaving enzyme.
However, the methods in the prior art frequently rely on the reformation or generation through homology recombination of an intact selectable or screenable marker gene.
WO06/105946 had also described a method for the exact exchange in plant cells and plants of a target DNA sequence for a DNA sequence of interest through homologous recombination, whereby the selectable or screenable marker used during the homologous recombination phase for temporal selection of the gene replacement events can subsequently be removed without leaving a foot-print and without resorting to in vitro culture during the removal step, employing the therein described method for the removal of a selected DNA by microspore specific expression of a double stranded break inducing rare cleaving endonuclease.
Therefore, there remains a need for alternative or improved methods which would allow targeted exchange of virtually any target DNA sequence by a replacement DNA. These and other problems are solved as described hereinafter in the different detailed embodiments of the invention, as well as in the claims.